RESUMO
The ongoing evolution of Ebolaviruses poses significant challenges to the development of immunodiagnostics for detecting emergent viral variants. There is a critical need for the discovery of monoclonal antibodies with distinct affinities and specificities for different Ebolaviruses. We developed an efficient technology for the rapid discovery of a plethora of antigen-specific monoclonal antibodies from immunized animals by mining the VH:VL paired antibody repertoire encoded by highly expanded B cells in the draining popliteal lymph node (PLN). This approach requires neither screening nor selection for antigen-binding. Specifically we show that mouse immunization with Ebola VLPs gives rise to a highly polarized antibody repertoire in CD138(+) antibody-secreting cells within the PLN. All highly expanded antibody clones (7/7 distinct clones/animal) were expressed recombinantly, and shown to recognize the VLPs used for immunization. Using this approach we obtained diverse panels of antibodies including: (i) antibodies with high affinity towards GP; (ii) antibodies which bound Ebola VLP Kissidougou-C15, the strain circulating in the recent West African outbreak; (iii) non-GP binding antibodies that recognize wild type Sudan or Bundibugyo viruses that have 39% and 37% sequence divergence from Ebola virus, respectively and (iv) antibodies to the Reston virus GP for which no antibodies have been reported.
Assuntos
Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Animais , Anticorpos Antivirais/genética , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Reações Cruzadas , Modelos Animais de Doenças , Epitopos/genética , Epitopos/imunologia , Doença pelo Vírus Ebola/genética , Humanos , Imunização , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Linfonodos/imunologia , Camundongos , Fenótipo , Ligação Proteica/imunologiaRESUMO
The Chinese hamster dihydrofolate reductase (DHFR) DNA replication initiation region, the 5.8 kb ori-beta, can function as a DNA replicator at random ectopic chromosomal sites in hamster cells. We report a detailed genetic analysis of the DiNucleotide Repeat (DNR) element, one of several sequence elements necessary for ectopic ori-beta activity. Deletions within ori-beta identified a 132 bp core region within the DNR element, consisting mainly of dinucleotide repeats, and a downstream region that are required for ori-beta initiation activity at non-specific ectopic sites in hamster cells. Replacement of the DNR element with Xenopus or mouse transcriptional elements from rDNA genes restored full levels of initiation activity, but replacement with a nucleosome positioning element or a viral intron sequence did not. The requirement for the DNR element and three other ori-beta sequence elements was conserved when ori-beta activity was tested at either random sites or at a single specific ectopic chromosomal site in human cells. These results confirm the importance of specific cis-acting elements in directing the initiation of DNA replication in mammalian cells, and provide new evidence that transcriptional elements can functionally substitute for one of these elements in ori-beta.
Assuntos
Origem de Replicação , Tetra-Hidrofolato Desidrogenase/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Cromossomos Humanos/genética , Cricetinae , Cricetulus , Primers do DNA/genética , Repetições de Dinucleotídeos , Células HeLa , Humanos , Camundongos , Fatores de Transcrição/metabolismo , Transfecção , XenopusRESUMO
A small DNA fragment containing the high-frequency initiation region (IR) ori-beta from the hamster dihydrofolate reductase locus functions as an independent replicator in ectopic locations in both hamster and human cells. Conversely, a fragment of the human lamin B2 locus containing the previously mapped IR serves as an independent replicator at ectopic chromosomal sites in hamster cells. At least four defined sequence elements are specifically required for full activity of ectopic ori-beta in hamster cells. These include an AT-rich element, a 4-bp sequence located within the mapped IR, a region of intrinsically bent DNA located between these two elements, and a RIP60 protein binding site adjacent to the bent region. The ori-beta AT-rich element is critical for initiation activity in human, as well as hamster, cells and can be functionally substituted for by an AT-rich region from the human lamin B2 IR that differs in nucleotide sequence and length. Taken together, the results demonstrate that two mammalian replicators can be activated at ectopic sites in chromosomes of another mammal and lead us to speculate that they may share functionally related elements.
